Hemolytic active proteins and genes encoding the same

ABSTRACT

Novel proteins providing the new approach to development of the drugs and pesticides with the use or application of a hemolytic activity, and novel proteins having the following properties and the genes encoding thereof are provided: 
     (1) having hemolytic activity; 
     (2) having a molecular weight of about 50,000 Da (determined by SDS gel electrophoresis); 
     (3) having an amino acid sequence represented by any of SEQ ID NO 1 to SEQ ID NO 3 as a partial amino acid sequence; and 
     (4) having an amino acid sequence represented by SEQ ID NO 5 as the full amino acid sequence.

TECHNICAL FIELD

The present invention relates to proteins having a hemolytic activityand genes encoding thereof. More specifically, the present inventionrelates to novel proteins having the hemolytic activity, a process forproducing and the use of the same.

BACKGROUND ART

The sting injury by the jellyfish in sea bathing has occurred in variousparts of the world. The sting injury by Carybdea rastonii or Physaliaphysalis has also occurred frequently in Japan every year in the seasonof sea bathing of the summertime. The degree of the symptom by stingdiffers by species of a jellyfish and the individual differences ofpatients. The first symptom is dermotoses, such as pain, flare, papule,vesicle and so on in the sting site. In a serious illness, patients maydie with generating of hemorrhagic maculae and the necrosis, and alsoconstitutional symptom, such as headache, high fever, nausea, dyspnea,and the fluctuation of a pulse.

Although such sting injury is occurring frequently, the determinationand pharmacological properties of the toxic components of jellyfish havenot been studied intensively.

Therefore, the development of medicines for treatment of the sting bythe jellyfish is hardly performed before the present invention.

The studies on the toxic components of Carybdea rastonii have reportedby Sato et al., and they found that there are some active substanceshaving physiological activities, such as hemolysis, plateletagglutination, mast cell degranulation, the vessel smoothness musclecontraction, the dermal necrosis, the heart poison and the fatality inthe crude extract fractions from the freeze-dried tentacle of Carybdearastonii. They also examined on the platelet agglutination effect andvessel smoothness muscle contraction effect of the toxic component(Akihiko Sato, “Research on the toxic component of Carybdea rastonii”,The Journal of the Ochanomizu Medico-dental Society, vol. 33, No. 2,131-151, June, 1985).

On the one hand, since the poison from the nematocyst of a jellyfish wasa non-dialyzable high polymer and deactivated by treatment with acid oralkali, or by heating processing, organic solvent processing, proteaseprocessing, etc., it was thought that the main components of this poisonwere proteins.

Moreover, the purification of the protein toxin derived from a jellyfishhas also been tried; however, the isolation and the purification of theactive components maintaining the hemolytic activity were not performedsince the toxin of a jellyfish itself was very easy to be deactivated.Therefore, the physical and chemical properties of the toxin fromjellyfish were not known up to now.

The detailed studies on the toxic component of a jellyfish is veryimportant for the development of drugs applying their variousphysiological activities, in particular, specific hemolytic activity andthe platelet agglutination effect.

Therefore, the problems to be solved by the present invention isproviding an approach to development of the drugs for treatment of thesting injury by the jellyfish by means of isolating the proteins orpeptides having as potent hemolytic activity as possible, in the statewhere the physiologic activity is retained. The present inventionfurther provides the approach to study similarities on embryology orstructure, and the species specificity of the protein having hemolyticactivity to evaluate the structure-activity relationship thereof.

DISCLOSURE OF THE INVENTION

The inventors extensively performed the research for isolating theproteins having the hemolytic activity from the nematocyst of Carybdearastonii using the hemolytic activity as the parameter, while retainingthese hemolytic activities. As the result, they found out the processfor isolating and purifying the proteins retaining hemolytic activities,and found that the protein from Carybdea rastonii had the partialchemical structure consisting the following amino acid sequences(1)-(3), and the molecular weight of about 50,000 Da (determined by SDSgel electrophoresis).

Amino acid sequence (1):

Gly-Glu-Ile-Gln-Thr-Lys-Pro-Asp-Arg-Val-Gly-Gln-Ala-Thr (SEQ ID NO:1)

Amino acid sequence (2):

Gly-Asn-Ala-Glu-His-Val-Ala-Ser-Ala-Val-Glu-Asn-Ala-Asn-Arg-Val-Asn-Lys(SEQ ID NO:2)

Amino acid sequence (3):

Met-Ser-Asp-Gly-Phe-Tyr-Thr-Met-Glu-Asn-Ser-Asp-Arg-Arg-Lys (SEQ IDNO:3)

(wherein, an amino acid residue is written by the 3 letters notationdefined by IUPAC and IUB)

Furthermore, they prepared the primers based on their partial chemicalstructures of the protein, and analyzed the gene sequence of about 1,000base pair of said protein by conducting the RT-PCR on the total RNAprepared from the tentacle of Carybdea rastonii by using these primers.Consequently, they further determined the full primary amino acidsequence of the hemolytic active protein of Carybdea rastonii by meansof analyzing the gene sequence at the 5′-end and 3′-end using the 5′RACE method and 3′ RACE method.

Therefore, one embodiment of the present invention provides the specificprotein having above-mentioned physiological, physical and chemicalproperties and represented by the amino acid (SEQ ID NO:5) or the aminoacid sequence thereof partially modified by the deletion or substitutionof amino acid, and/or the amino acid sequence thereof partially modifiedby the deletion or substitution of amino acid further one or more aminoacids are added.

Another embodiment of the present invention also provides the processfor preparing such proteins.

Furthermore, another embodiment provides the gene encoding suchproteins, the process for preparing the specific proteins using thegene, and the drugs or the pesticides using the same.

The present invention further provides the pharmaceutical compositionsor the pesticides containing the proteins using these properties,particularly, the pharmaceutical compositions having the plateletagglutination effect etc.

Moreover, since a specific antibody can also be obtained from thishemolytic active protein according to a conventional method (CellTechnology, separate volume, “Experimental protocol of antipeptideantibody”, Shujunsha Co.), the present invention also provides thepharmaceutical compositions containing said antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

The isolation and purification of the proteins having the specificphysiological activity provided by the present invention canspecifically be performed as follows. For example, the ultrasonicationof the nematocyst of Carybdea rastonii is carried out in phosphoric acidbuffer solution, and then supernatants are collected by the centrifugalseparation to obtain a crude extract. The object proteins can beseparated and purified by subjecting this crude extract to ion exchangehigh performance liquid chromatography using TSK-GEL (Toso Co.), and thegel filtration high performance liquid chromatography with Superdex-75(Pharmacia Co.).

The structure of the protein provided according to the present inventionobtained in this way can be determined by combining the analysisprocedure of the amino acid sequence by the selective degradation usingthe enzyme, and the analysis procedure of a gene sequence using the PCRmethod etc. For example, the amino acid sequence can be determined byprocessing the protein separated and purified as mentioned above with alysylendopeptidase, fractionating the fragment using a high performanceliquid chromatography, and analyzing it using an amino acid sequenceretc. Next, the gene sequence of the proteins can be determined by RT-PCRmethod etc. using the primers prepared on the basis of the amino acidsequence. Finally, the full primary amino acid sequence of the proteinscan be clarified by determining the amino acid sequence on the basis ofthe gene sequence.

It was confirmed by such analysis that the protein provided according tothe present invention has the molecular weight of about 50,000 Da(measured by SDS gel electrophoresis), and the partial amino acidsequences have the above-mentioned amino acid sequences (1) to (3).

As a result of homology search on the partial amino acid sequences, thehomology between the protein of the present invention and the knownproteins was very low. Therefore, it was suggested that the protein ofthe present invention having the hemolytic activity is completely novelprotein, which is not similar to the known proteins.

Next, the determination of the gene sequence of about 1,000 base pairsby performing RT-PCR to total RNA prepared from the tentacle of Carybdearastonii using the primers prepared on the basis of the partial aminoacid sequence, and the determination of the gene sequences of the 5′-endand the 3′-end using the 5′ RACE method and 3′ RACE method wereperformed. Consequently, it is concluded that the hemolytic activeprotein of Carybdea rastonii has the full primary amino acid sequencerepresented by (SEQ ID NO:5) and the gene encoding thereof has the basesequence represented by (SEQ ID NO:4).

The result of the homology search on these full primary amino acidsequences exhibited that the homology between the protein and the knownproteins was low.

The method for preparing the specific protein of the present inventionby separation and purification is characterized in retaining thehemolytic activity. For example, the separation and the purification inthe state of retaining such hemolytic activity are attained byperforming the processing such as ultrasonication using theabove-mentioned phosphoric acid buffer solution or various highperformance liquid chromatography in 10 mM phosphoric acid buffersolution (pH 6.0) containing above 0.1 M NaCl, preferably above 0.3 M,and more preferably above 0.5 M, at below 10° C., preferably below 5° C.

Therefore, the present invention also provides the method for preparingthe protein by extracting and purifying them from the nematocyst of theCarybdea rastonii in the state of retaining the physiological activity.

The specific protein of the present invention also can be prepared bythe gene recombination method. Preparation by the gene recombinationmethod can be performed according to a conventional method. For example,it can be obtained by preparing the vector integrated with the generepresented by (SEQ ID NO:4), transforming a host cell by the vector,incubating or growing the host cell, and isolating and purifying theproteins having hemolytic activity of interest from the host cell orculture solution.

Since the protein provided according to the present invention has ahemolytic activity, for example, it may be used for the medicamentshaving the platelet agglutination effect and for the reagents forresearch on a hemolysis. Furthermore, it provides the new approach forthe development of drugs, such as a drug for treating the sting by thejellyfish, and development of pesticides, such as an insecticide, usingthe hemolytic activity.

EXAMPLES

The present invention will be described in detail with reference to thefollowing examples; however, the present invention is not limited to theexamples.

Example 1 1) Extraction of the Nematocyst of Carybdea rastonii

200 mg of the ematocyst of the Carybdea rastoniiobtained on the Miurapeninsula, Kanagawa, Japan and cryopreservated at −80° C. was immersedin 8 ml of 10 mM phosphoric acid buffer solution (pH 6.0), and treatedfor 15 minutes by the ultrasonic wave (ultrasonic cleaner VS150, IuchiCo.). The supernatant fluids were collected by centrifugal separation(3,000 rpm, for 20 minutes). This operation was performed 3 times intotal. Furthermore, the same extraction operation was repeated 3 timeswith 8 ml of 10 mM phosphoric acid buffer solutions (pH 6.0) containing1 M NaCl, and then all the supernatant fluids were collected. After theextraction operation, ion exchange HPLC (high performance liquidchromatography) of the following purification step was immediatelyperformed.

2) The Purification by Ion Exchange HPLC (Column: TSK-GELCM650S, ColumnSize: 20×220 mm)

The above-mentioned column was equilibrated with 10 mM phosphoric acidbuffer solution (pH 6.0) containing 0.3 M NaCl. After the equilibration,the supernatant fluids obtained by extraction in the operation of theabove-mentioned 1) were combined and diluted with 10 mM phosphoric acidbuffer solution (pH 6.0) to 4 times. The solution was loaded onto theabove-mentioned column at a flow rate of the 3 ml/min. The column waswashed with 100 ml of 10 mM phosphoric acid buffer solutions (pH 6.0)after the sample application. The elution was carried out by the 60minutes gradient in 0 to 0.7 M NaCl concentration (in 10 mM phosphoricacid buffer solution: pH 6.0). Hemolytic activity was showed in manyfractions eluting between 45 and 65 minutes after start of the gradient.In addition, hemolytic activity was examined about the hemolytic effectto sheep hemocytes (see the after-mentioned example 2).

3) The Purification by Ion Exchange HPLC (Column: TSK-GEL CM5PW, ColumnSize: 7.5×75 mm)

The above-mentioned column was well equilibrated with 10 mM phosphoricacid buffer solution (pH 6.0) containing 0.3 M NaCl. The hemolyticactive fractions obtained by purifying operation of the above-mentioned2) were diluted with 10 mM phosphoric acid buffer solution (pH 6.0) to 4times. The solution was loaded onto the above-mentioned column at theflow rate of 2 ml/min. The column was washed with 30 ml of 10 mMphosphoric acid buffer solutions (pH 6.0) after the sample application.After washing, the elution was performed by the 60 min gradient in 0 to0.8 M NaCl concentration (in 10 mM phosphoric acid buffer solution: pH6.0). Fractions having hemolytic activity were eluted between 25 and 35minutes after start of the gradient, and each fraction was applied toSDS-PAGE. The separating condition of the active component was verified,and the portions separated well were collected and used in the nextstep. On the contrary, the portions not separated were further performedby chromatography to complete the separation of the active component.

4) Concentration of the Hemolytic Active Component by Ion Exchange HPLC(Column: TSK-GEL CM5PW, Column Size: 7.5×75 mm)

The column was well equilibrated with 10 mM phosphoric acid buffersolution (pH 6.0) containing 0.3 M NaCl. The hemolytic active fractionsobtained by purifying operation of above-mentioned 3) were diluted with10 mM phosphoric acid buffer solution (pH 6.0) to 4 times. The solutionwas loaded onto the above-mentioned column at the flow rate of 2 ml/min.The column was washed with 30 ml of 10 mM phosphoric acid buffersolutions (pH 6.0) after the sample application. After washing, 10 mMphosphoric acid buffer solution (pH 6.0) containing 0.8 M NaCl was thenrinsed and the sample adhered into the column was allowed to elute. Inabout 5 minutes after exchange of the solvent, the portion of thehemolytic active component condensed and eluted at a stretch wascollected.

5) The Purification by Gel Filtration HPLC (Column: Superdex-75, ColumnSize: 16×600 mm)

Every 0.5-1.0 ml of the sample condensed by ion exchange HPLC wasapplied to the above-mentioned column equilibrated with 10 mM phosphoricacid buffer solution (pH 6.0) containing 0.8 M NaCl, and allowed toelute at the flow rate of 1 ml/min. Potent hemolytic activity was foundout in the fraction eluting between 50 and 60 minutes after injection ofthe sample. After confirming the separating condition by SDS PAGE, theprotein of the present invention, a hemolytic toxin, was separated bycollecting the active fractions (about 1 μg).

Example 2 Measurement of the Hemolytic Activity

Measurement of the hemolytic activity in each purification step in theabove-mentioned Example 1 and measurement of the hemolytic activity ofthe protein of the present invention finally obtained were performed asfollows.

1) Method

Hemolytic activity was measured by hemolysis to a sheep erythrocyte.That is, every 200 μl of PBS(+) buffer solution containing 0.8% of sheeperythrocyte was put into the microwell plates of 96 wells (round bottomtype). 10 μl of the solution dissolved the fraction obtained in eachpurification step of the above-mentioned Example 1 in 10 mM phosphoricacid buffer solution (pH 6.0) was added to the plate. It was allowed tostand at room temperature for 3 hours, and the hemolytic condition ofthe sheep erythrocyte of each plate was observed. In addition, thepresence or absence of the retention of the hemolytic activity wasdetermined by whether the fraction obtained in each purification stepexhibits a perfect hemolysis.

2) Results

2-1) The fraction obtained in each purification step of theabove-mentioned Example 1 exhibited the perfect hemolysis to the sheeperythrocyte, and therefore, it became clear that it retains thehemolytic activity.

2-2) Moreover, the protein of the present invention having the hemolyticactivity finally obtained by purification operation of theabove-mentioned 5) in Example 1 caused the perfect hemolysis to thesheep erythrocyte in the concentration below 100 ng/ml (about 2 nM).

Example 3 Determination of the Molecular Weight and the PartialStructure on the Proteins

3-1) Determination of the Molecular Weight

The single band visualized by applying the protein of the presentinvention having the hemolytic activity obtained by purificationoperation of 5) in Example 1 to SDS gel electrophoresis (SDS-PAGE)according to the conventional method was compared with the proteinmolecular-weight marker (Pharmacia Co.). As the result, it wasidentified that the molecular weight of the protein of the presentinvention are about 50,000 Da.

3-2) Decomposition With the Lysylendopeptidase

The protein was decomposed by adding 3 pM of Achromobacter Protease I(derived from Achromobacter lyticus M497-1: Takara Shuzo Co.) to 10 μgof protein according to the present invention having the hemolyticactivity obtained by purification operation of the above-mentioned 5) inExample 1, and incubating in 10 mM of Tris-HCl buffer solution (pH 9.0)at 30° C. for 20 hours. The protein digested with the enzyme was appliedto the high performance liquid chromatography (column: Bakerbond widepore ODS), and separated with the 60 min gradient in 10 to 62% ofacetonitrile concentration (in water containing 0.1% of trifluoroaceticacid) at the flow rate of 0.7 ml/min. Consequently, three peptidefragments eluting respectively at a retention time 19, 23 and 27 minuteswere obtained.

3-3) Determination of the Amino Acid Sequence of Each Fragments by theAmino Acid Sequencer

The amino acid sequence of three peptide fragments obtained as mentionedabove was determined according to the conventional method using ShimadzuPSQ-1 protein sequencer (Shimadzu Co.).

As the result, three fragments have the following amino acid sequences(1)-(3), respectively:

Amino acid sequence (1):

Gly-Glu-Ile-Gln-Thr-Lys-Pro-Asp-Arg-Val-Gly-Gln-Ala-Thr (SEQ ID NO:1)

Amino acid sequence (2):

Gly-Asn-Ala-Glu-His-Val-Ala-Ser-Ala-Val-Glu-Asn-Ala-Asn-Arg-Val-Asn-Lys(SEQ ID NO:2)

Amino acid sequence (3):

Met-Ser-Asp-Gly-Phe-Tyr-Thr-Met-Glu-Asn-Ser-Asp-Arg-Arg-Lys (SEQ IDNO:3)

(wherein, an amino acid residue is written by the 3 letters notationdefined by IUPAC and IUB).

The homology search about each fragment with which the amino acidsequence was determined as mentioned above exhibited that the homologybetween these fragments and the known proteins was very low. Therefore,it was suggested that the specific protein of the present inventionfractionated from the nematocyst of Carybdea rastonii while retainingthe hemolytic activity is completely novel protein.

Example 4 Determination of the Full Amino Acid Sequence of the Proteinand the Gene Encoding the Amino Acids

4-1) Preparation of Total RNA of Carybdea rastonii

The tentacle (about 0.5 g in wet weights) of Carybdea rastonii wascrushed in the liquid nitrogen, and homogenized in 5 ml TRIzol(registered trademark) reagent (GIBCO BRL Co.). To this mixture wasadded 1 ml of chloroform, and the mixture was agitated, and centrifugedwith the cooling centrifuge (Sakuma Co.) [13,000 rpm, for 15 minutes, at4° C.]. The upper aqueous layer was fractionated, and to this solutionwas added 2.5 ml of isopropanol, then, the mixture was allowed to standat room temperature for 10 minutes. The supernatant fluid was removedafter the centrifugal separation (13,000 rpm, for 10 minutes, at 4° C.)using the cooling centrifuge, and then 5 ml of 75% ethanol was added theresidue. The supernatant fluid was removed after the centrifuge (10,000rpm, for 5 minutes, at 4° C.) to obtain the residue, then, theair-drying of the residue was performed for about 10 minutes. 100 μl ofRNase-free water was added to the resulting residue, and the mixture wasincubated for 10 minutes at 60° C. to lyse RNA. About 0.5 mg of totalRNA was obtained according to the above-mentioned method.

4-2) Cloning of a Partial cDNA

On the basis of amino acid sequence (1), amino acid sequence (2) andamino acid sequence (3), the following degenerate primers were designedand synthesized by the conventional method:

7-F; GAR ATH CAR ACI AAR CCI G (SEQ ID NO:6)

7-R; CIG GYT TIG TYT GDA TYT C (SEQ ID NO:7)

12-F; GCI GTI GAR AAY GCI AAY MG (SEQ ID NO:8)

12-R; CKR TTI GCR TTY TCI ACI GC (SEQ ID NO:9)

14-1-F; GAY GGI TTY TAY ACI ATG G (SEQ ID NO:10)

14-1-R; CCA TIG TRT ARA AIC CRT C (SEQ ID NO:11)

12-2-F; GAY GGI TTY TAY ACI ATG GAR AA (SEQ ID NO:12)

12-2-R; TTY TCC ATI GTR TAR AAI CCR TC (SEQ ID NO:13)

(wherein, the above-mentioned alphabetic character was written based onthe “Nucleotide Abbreviation List” (Cell Technology, separate volume,“Biotechnology Experiment Illustrated”: Shujunsha Co.).

Next, according to the following procedure, single-strand cDNA wassynthesized using SUPERSCRIPT (registered trademark) PreamplificationSystem for 1st-Strand cDNA Synthesis. That is, 1 μg of total RNA,oligo(dT)₁₂₋₁₈, and DEPC-treated water were mixed, and the mixture wasallowed to stand for 10 minutes at 70° C. Then, PCR buffer, 25 mM MgCl₂,10 mM dNTP mix, and 0.1 M DTT were added to this mixture, and theresulting mixture was pre-incubated for 5 minutes at 42° C. SuperscriptII RT (200 units/μl) was added to this mixture, and the mixture wasincubated for 50 minutes at 42° C. and for 15 minutes at 70° C. TheRNase H was added to the mixture, and then, the resulting mixture wasincubated for 20 minutes at 37° C. to obtain 1st-strand cDNA.

Subsequently, according to the following conditions, PCR was performedusing GeneAmp PCR System 2400 thermal cycler (Perkin-Elmer Co.). Thatis, 1st-strand cDNA, PCR buffer, dNTP mix, primer 1 and primer 2(wherein, primer 1 and primer 2 are any eight above-mentioned primers.),TaKaRa Ex Taq (registered trademark, Takara Shuzo Co.), and water weremixed. The reaction was performed by heating the mixture at 94° C. for 5minutes and repeating 3 cycles of 30 seconds at 94° C., 30 seconds at45° C. and 2 minutes at 72° C., and 27 cycles of 30 seconds at 94° C.,30 seconds at 55° C. and 2 minutes at 72° C. The reactant was thentreated for 5 minutes at 72° C.

The obtained reaction solution was electrophoresed on 0.8% agarose gelto confirm the amplified PCR products in the combination of 7-F and12-R, 7-F and 14-1-R, 7-F and 14-2-R, 12-F and 14-1-R, and 12-F and14-2-R. The sizes of each PCR product were about 600 bp, 1,000 bp, 1,000bp, 400 bp, and 400 bp, respectively.

4-3) Sequencing of the Partial cDNA

Each PCR product was inserted into TA cloning vector pCR2.1 (InvitrogeneCo.), and the recombinant was transformed to the Escherichia coli JM109.The transformant was cultured on LB (containing 50 μg/μl of ampicillin)agar medium. According to the following conditions, colony PCR wasperformed to the colonies obtained as a template using the M13 universalprimer. The strain of Escherichia coli, PCR buffer, dNTP mix, M13 FWprimer, M13 RV primer, TaKaRa Ex Taq (registered trademark, Takara ShuzoCo.), and water were mixed. The reaction was performed by heating themixture at 90° C. for 10 minutes and repeating 30 cycles of 30 secondsat 94° C., 30 seconds at 55° C. and 2 minutes at 72° C., and thenheating at 72° C. for 5 minutes. The reaction solution waselectrophoresed on 0.8% agarose gel and the target colony PCR productwas purified on the spin column of MicroSpin (registered trademark)S-400 (Amersham Pharmacia Co.). Then, the sequencing of the obtainedproduct was conducted using ABI PRISM 310 Genetic Analyzer (AppliedBiosystems Co.).

The obtained sequence was analyzed using gene analysis softwareGENETYX-MAC (Software Development Co.). As the result, the partial cDNAsequence of about 1000 bp was analyzed, and each partial structure ofamino acid sequence (1), amino acid sequence (2) and amino acid sequence(3) was determined to locate in this turn from N terminal of theprotein.

4-4) Sequencing of the Full-length cDNA

Following primers were synthesized based on the base sequence of thepartial cDNA:

5′-RACE-4R; GCT CTA TCA ATA ACG GCA GC (SEQ ID NO:14)

5′-RACE-5R; TGT CTT TGG ATG GCC TCA TC (SEQ ID NO:15)

5′-RACE-6R; GAT ACT TAG GTC GCT ATC CG (SEQ ID NO:16)

3′-RACE-1F; GTT CAG AGG CTG TTC TAA CG (SEQ ID NO:17)

3′-RACE-2F; ATG TCT GAC GGC TTC TAC AC (SEQ ID NO:18)

Next, according to the following procedure, 5′ RACE and 3′ RACE wereperformed using 5′/3′ RACE Kit (Boehringer Mannheim Co.).

(a) 5′ RACE

1 μg of total RNA, cDNA synthesis buffer, DNTP mix, 5′-RACE-6R, AMVreverse transcriptase, and DEPC-treated water were mixed, and themixture was incubated for 60 minutes at 55C and for 10 minutes at 65° C.to obtain 1st-strand cDNA.

Next, 1st-strand cDNA thus obtained was purified on the spin column,then, reaction buffer and 2mM DATP were added to the 1st-strand cDNA,and the mixture was allowed to stand for 3 minutes at 94° C. Terminaltransferase (10 units/μl) was added to the mixture, and the resultingmixture was incubated for 20 minutes at 37° C. After the incubation,1st-strand cDNA, PCR buffer, dNTP mix, 5′-RACE-5R, oligo(dT)-anchorprimer, and water were added to the above mixture. The reaction wasperformed by heating the mixture at 94° C. for 5 minutes and repeating30 cycles of 30 seconds at 94° C., 30 seconds at 55° C. and 1 minute at72° C., and then heating at 72° C. for 5 minutes. Consequently, thenested-PCR was performed to the 1st-PCR product as a template using thecombination of 5′-RACE-4R and PCR anchor primer under the same conditionas 1st-PCR.

The 1st-PCR product and the nested-PCR product were electrophoresed on1.5% agarose gel to confirm the band of about 500 bp. This nested-PCRproduct was inserted into TA cloning vector, and the sequencing wasperformed according to the determination of the base sequence of CDNAdescribed in the above-mentioned 4-3), then the sequence was analyzed.

(b) 3′ RACE

1 μg of total RNA, cDNA synthesis buffer, DNTP mix, oligo(dT)-anchorprimer, AMV reverse transcriptase, and DEPC-treated water were mixed,and the mixture was incubated for 60 minutes at 55° C. Subsequently, thereactant was treated for 10 minutes at 65° C. to obtain 1st-strand cDNA.

Next, 1st-PCR thus obtained was performed under the following condition.1st-strand cDNA, PCR buffer, dNTP mix, 3′-RACE-lF, PCR anchor primer,TaKaRa Ex Taq (registered trademark, Takara Shuzo Co.), and water weremixed. The reaction was performed by heating the mixture at 94° C. for 5minutes and repeating 30 cycles of 30 seconds at 94° C., 30 seconds at55° C. and 2 minutes at 72° C., and then heating at 72° C. for 5minutes. The nested-PCR was performed to the 1st-PCR product as atemplate using the combination of 3′-RACE-2F and PCR anchor primer underthe same condition as 1st-PCR.

The 1st-PCR product and the nested-PCR product were electrophoresed on1.5% agarose gel to confirm the band of about 600 bp. The nested-PCRproduct was inserted into TA cloning vector, the sequencing wasperformed according to the determination of the base sequence of cDNAdescribed in the above-mentioned 4-3), and the sequence was analyzed.

As a result, the size (1610 bp) and the sequence of cDNA encoding thenovel hemolytic active protein of Carybdea rastonii, and the number(450aa) and the sequence of amino acid of the protein became clear. Thatis, the hemolytic active protein of Carybdea rastonii had the amino acidsequence represented by (SEQ ID NO:5), and the gene encoding thereof hadthe base sequence represented by (SEQ ID NO:4).

The amino acid sequence (1) (SEQ ID NO:1), the amino acid sequence (SEQID NO:2), and the amino acid sequence (3) (SEQ ID NO:3) corresponded tothe amino acid number 56-69 of (SEQ ID NO:5), the amino acid number250-267 of (SEQ ID NO:5), and the amino acid number 363-377 of (SEQ IDNO:5), respectively. Furthermore, it was confirmed that the poly Asequence exists after the nucleotide number 1600 of (SEQ ID NO:4).

The novel protein of the present invention obtained as mentioned aboveis the specific protein having the following physiological activity, andphysical and chemical property, as indicated by the example:

(a) having hemolytic activity;

(b) having a molecular weight of about 50,000 Da (determined by SDS gelelectrophoresis);

(c) having the amino acid sequences 1 to 3 described above as a partialamino acid sequence; and

(d) having the amino acid sequence represented by SEQ ID NO 5 as thefull amino acid sequence.

Industrial Applicability

Since the protein having the hemolytic activity derived from thenematocyst of Carybdea rastonii provided according to the presentinvention is a novel protein which is not similar to known protein, as aresult of the homology search on the partial amino acid sequence and thefull primary amino acid sequences, it is useful as a biochemical reagentfor example, elucidating the mechanism of a hemolysis etc.

It also provides the new approach directed to development of drugs, suchas the medicine for treating the sting by the jellyfish, on the basis ofstudy of correlation of the structural activity in a molecular level,and the antibody on the protein or the partial peptide, etc.Furthermore, it is useful as the drugs having a platelet agglutinationeffect etc., and pesticides using a hemolytic activity.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 18 <210> SEQ ID NO 1 <211>LENGTH: 14 <212> TYPE: PRT <213> ORGANISM: Carybdea rastonii <220>FEATURE: <223> OTHER INFORMATION: This amino acid residue sequencecorresponds to amino acid residue positions 56-69 of SEQ ID NO:5. <400>SEQUENCE: 1 Gly Glu Ile Gln Thr Lys Pro Asp Arg Val Gly Gln Ala Thr 1 510 <210> SEQ ID NO 2 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM:Carybdea rastonii <220> FEATURE: <223> OTHER INFORMATION: This aminoacid residue sequence corresponds to amino acid residue positions250-267 of SEQ ID NO:5. <400> SEQUENCE: 2 Gly Asn Ala Glu His Val AlaSer Ala Val Glu Asn Ala Asn Arg Val 1 5 10 15 Asn Lys <210> SEQ ID NO 3<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Carybdea rastonii <220>FEATURE: <223> OTHER INFORMATION: This amino acid residue sequencecorresponds to amino acid residue positions 363-377 of SEQ ID NO:5.<400> SEQUENCE: 3 Met Ser Asp Gly Phe Tyr Thr Met Glu Asn Ser Asp ArgArg Lys 1 5 10 15 <210> SEQ ID NO 4 <211> LENGTH: 1610 <212> TYPE: DNA<213> ORGANISM: Carybdea rastonii <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (28)..(1380) <221> NAME/KEY: protein_bind <222>LOCATION: (1381)..(1610) <400> SEQUENCE: 4 gcacaagcga cttggtgaag gagcaccatg att ctg aaa cat ctt cct tgg ctc 54 Met Ile Leu Lys His Leu Pro TrpLeu 1 5 ttt att gtc ctt gca att act tct gca aaa cat ggc aaa cgc tct gat102 Phe Ile Val Leu Ala Ile Thr Ser Ala Lys His Gly Lys Arg Ser Asp 1015 20 25 gtc aat tct tta ctt act aag gta gaa act gcc tta aaa gaa gct tct150 Val Asn Ser Leu Leu Thr Lys Val Glu Thr Ala Leu Lys Glu Ala Ser 3035 40 ggt agc aac gag gct gct ctt gag gct tta gag ggc tta aaa gga gag198 Gly Ser Asn Glu Ala Ala Leu Glu Ala Leu Glu Gly Leu Lys Gly Glu 4550 55 atc cag aca aaa cca gac cga gtt gga caa gcc aca aaa atc ctt gga246 Ile Gln Thr Lys Pro Asp Arg Val Gly Gln Ala Thr Lys Ile Leu Gly 6065 70 tct gtc gga tca gct cta gga aaa tta aat tct gga gat gca acc aaa294 Ser Val Gly Ser Ala Leu Gly Lys Leu Asn Ser Gly Asp Ala Thr Lys 7580 85 atc att tct ggt tgc ctc gac att gtt gca gga att gca aca act ttt342 Ile Ile Ser Gly Cys Leu Asp Ile Val Ala Gly Ile Ala Thr Thr Phe 9095 100 105 gga ggc cct gtc ggg atg gga atc gga gcc gta gct tct ttt gtttct 390 Gly Gly Pro Val Gly Met Gly Ile Gly Ala Val Ala Ser Phe Val Ser110 115 120 tca att cta tca ttg ttt act gga agc tca gca aag aac tca gttgct 438 Ser Ile Leu Ser Leu Phe Thr Gly Ser Ser Ala Lys Asn Ser Val Ala125 130 135 gcc gtt att gat aga gct tta agc aag cat cgc gat gag gcc atccaa 486 Ala Val Ile Asp Arg Ala Leu Ser Lys His Arg Asp Glu Ala Ile Gln140 145 150 aga cat gca gca ggt gcc aag aga gat ttt gct gaa tca tct gcattc 534 Arg His Ala Ala Gly Ala Lys Arg Asp Phe Ala Glu Ser Ser Ala Phe155 160 165 att cag gtc atg aaa cag cag tcc aat ctt aca gat agc gac ctaagt 582 Ile Gln Val Met Lys Gln Gln Ser Asn Leu Thr Asp Ser Asp Leu Ser170 175 180 185 atc att gca gcg aat gtt cct gtt tat aaa ttt agt aat tttatc gga 630 Ile Ile Ala Ala Asn Val Pro Val Tyr Lys Phe Ser Asn Phe IleGly 190 195 200 cag ttg gag agc aga att tcc caa ggc gca gca act acc agtctt agc 678 Gln Leu Glu Ser Arg Ile Ser Gln Gly Ala Ala Thr Thr Ser LeuSer 205 210 215 gat gca aag aga gcc gtt gac ttc att ctg ctc tat tgt caactt gta 726 Asp Ala Lys Arg Ala Val Asp Phe Ile Leu Leu Tyr Cys Gln LeuVal 220 225 230 gtc atg aga gaa acc ttg ctg gtc gac ttg gct att ctc tacagg aaa 774 Val Met Arg Glu Thr Leu Leu Val Asp Leu Ala Ile Leu Tyr ArgLys 235 240 245 gga aat gca gaa cac gtg gca agt gct gtg gaa aac gct aatagg gta 822 Gly Asn Ala Glu His Val Ala Ser Ala Val Glu Asn Ala Asn ArgVal 250 255 260 265 aac aaa gag cta gct gct gat acc cta gat ttt ctt cataaa ttg att 870 Asn Lys Glu Leu Ala Ala Asp Thr Leu Asp Phe Leu His LysLeu Ile 270 275 280 cct gaa caa gca ttg ata ggt gca gtt tat cat cca atttct gcc tct 918 Pro Glu Gln Ala Leu Ile Gly Ala Val Tyr His Pro Ile SerAla Ser 285 290 295 gaa act agc aaa gca ata tta aat tac acg aaa tac tttgga gtt cca 966 Glu Thr Ser Lys Ala Ile Leu Asn Tyr Thr Lys Tyr Phe GlyVal Pro 300 305 310 gat gtt ccc cgt cct att gga aac cgc aga tac aaa tttaca aat agt 1014 Asp Val Pro Arg Pro Ile Gly Asn Arg Arg Tyr Lys Phe ThrAsn Ser 315 320 325 tac tgg aat acc tac agt ata tgc agt gag gct tac atggga aat tac 1062 Tyr Trp Asn Thr Tyr Ser Ile Cys Ser Glu Ala Tyr Met GlyAsn Tyr 330 335 340 345 atg ttc aga ggc tgt tct aac gtt cgg aat cca aatatc agg gta tcc 1110 Met Phe Arg Gly Cys Ser Asn Val Arg Asn Pro Asn IleArg Val Ser 350 355 360 aaa atg tct gat ggg ttt tac acc atg gag aat agcgat cgg agg aag 1158 Lys Met Ser Asp Gly Phe Tyr Thr Met Glu Asn Ser AspArg Arg Lys 365 370 375 ttg tat atc acc aag cat gac caa gga tgg gga tggggt act ttg gat 1206 Leu Tyr Ile Thr Lys His Asp Gln Gly Trp Gly Trp GlyThr Leu Asp 380 385 390 gag gat cca ggt gac caa ggc cat atg agg ttc attcct ttg aga cat 1254 Glu Asp Pro Gly Asp Gln Gly His Met Arg Phe Ile ProLeu Arg His 395 400 405 ggg aag tat atg gta agc tct aag agg tgg ccc aactgg ttc atg tat 1302 Gly Lys Tyr Met Val Ser Ser Lys Arg Trp Pro Asn TrpPhe Met Tyr 410 415 420 425 atg gaa tca agt gcc agt ggc tac att cgc agctgg gaa aat aat cca 1350 Met Glu Ser Ser Ala Ser Gly Tyr Ile Arg Ser TrpGlu Asn Asn Pro 430 435 440 gga cct caa gga cat tgg agt ata aca taattaaagagga atcaacaatg 1400 Gly Pro Gln Gly His Trp Ser Ile Thr 445 450tcccaaaggc atacgaatat aagacatcaa acgaatgcag tacttaaagt gcacacttgt 1460atttctacat aggatgtcgt catgaaagtc cataaaccat ccagcggact aatttcatat 1520taaacattaa tgtttcctta taatgcattt tcatgaaatc tctattgtga catttcaaga 1580ggatatgttt gaaagaaaca aaaaaaaaaa 1610 <210> SEQ ID NO 5 <211> LENGTH:450 <212> TYPE: PRT <213> ORGANISM: Carybdea rastonii <400> SEQUENCE: 5Met Ile Leu Lys His Leu Pro Trp Leu Phe Ile Val Leu Ala Ile Thr 1 5 1015 Ser Ala Lys His Gly Lys Arg Ser Asp Val Asn Ser Leu Leu Thr Lys 20 2530 Val Glu Thr Ala Leu Lys Glu Ala Ser Gly Ser Asn Glu Ala Ala Leu 35 4045 Glu Ala Leu Glu Gly Leu Lys Gly Glu Ile Gln Thr Lys Pro Asp Arg 50 5560 Val Gly Gln Ala Thr Lys Ile Leu Gly Ser Val Gly Ser Ala Leu Gly 65 7075 80 Lys Leu Asn Ser Gly Asp Ala Thr Lys Ile Ile Ser Gly Cys Leu Asp 8590 95 Ile Val Ala Gly Ile Ala Thr Thr Phe Gly Gly Pro Val Gly Met Gly100 105 110 Ile Gly Ala Val Ala Ser Phe Val Ser Ser Ile Leu Ser Leu PheThr 115 120 125 Gly Ser Ser Ala Lys Asn Ser Val Ala Ala Val Ile Asp ArgAla Leu 130 135 140 Ser Lys His Arg Asp Glu Ala Ile Gln Arg His Ala AlaGly Ala Lys 145 150 155 160 Arg Asp Phe Ala Glu Ser Ser Ala Phe Ile GlnVal Met Lys Gln Gln 165 170 175 Ser Asn Leu Thr Asp Ser Asp Leu Ser IleIle Ala Ala Asn Val Pro 180 185 190 Val Tyr Lys Phe Ser Asn Phe Ile GlyGln Leu Glu Ser Arg Ile Ser 195 200 205 Gln Gly Ala Ala Thr Thr Ser LeuSer Asp Ala Lys Arg Ala Val Asp 210 215 220 Phe Ile Leu Leu Tyr Cys GlnLeu Val Val Met Arg Glu Thr Leu Leu 225 230 235 240 Val Asp Leu Ala IleLeu Tyr Arg Lys Gly Asn Ala Glu His Val Ala 245 250 255 Ser Ala Val GluAsn Ala Asn Arg Val Asn Lys Glu Leu Ala Ala Asp 260 265 270 Thr Leu AspPhe Leu His Lys Leu Ile Pro Glu Gln Ala Leu Ile Gly 275 280 285 Ala ValTyr His Pro Ile Ser Ala Ser Glu Thr Ser Lys Ala Ile Leu 290 295 300 AsnTyr Thr Lys Tyr Phe Gly Val Pro Asp Val Pro Arg Pro Ile Gly 305 310 315320 Asn Arg Arg Tyr Lys Phe Thr Asn Ser Tyr Trp Asn Thr Tyr Ser Ile 325330 335 Cys Ser Glu Ala Tyr Met Gly Asn Tyr Met Phe Arg Gly Cys Ser Asn340 345 350 Val Arg Asn Pro Asn Ile Arg Val Ser Lys Met Ser Asp Gly PheTyr 355 360 365 Thr Met Glu Asn Ser Asp Arg Arg Lys Leu Tyr Ile Thr LysHis Asp 370 375 380 Gln Gly Trp Gly Trp Gly Thr Leu Asp Glu Asp Pro GlyAsp Gln Gly 385 390 395 400 His Met Arg Phe Ile Pro Leu Arg His Gly LysTyr Met Val Ser Ser 405 410 415 Lys Arg Trp Pro Asn Trp Phe Met Tyr MetGlu Ser Ser Ala Ser Gly 420 425 430 Tyr Ile Arg Ser Trp Glu Asn Asn ProGly Pro Gln Gly His Trp Ser 435 440 445 Ile Thr 450 <210> SEQ ID NO 6<211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Degenerate PCR primer, 7-F, used in the cloning of the partialcDNA of the hemolytic active protein of Carybdea rastonii <221>NAME/KEY: unsure <222> LOCATION: (12) <223> OTHER INFORMATION: n =inosine <221> NAME/KEY: unsure <222> LOCATION: (18) <223> OTHERINFORMATION: n = inosine <400> SEQUENCE: 6 garathcara cnaarccng 19 <210>SEQ ID NO 7 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (2) <223>OTHER INFORMATION: n = inosine <221> NAME/KEY: unsure <222> LOCATION:(8) <223> OTHER INFORMATION: n = inosine <223> OTHER INFORMATION:Description of Artificial Sequence: Degenerate PCR primer, 7-R, used inthe cloning of the partial cDNA of the hemolytic active protein ofCarybdea rastonii <400> SEQUENCE: 7 cnggyttngt ytgdatytc 19 <210> SEQ IDNO 8 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (3) <223>OTHER INFORMATION: n = inosine <221> NAME/KEY: unsure <222> LOCATION:(6) <223> OTHER INFORMATION: n = inosine <221> NAME/KEY: unsure <222>LOCATION: (15) <223> OTHER INFORMATION: n = inosine <223> OTHERINFORMATION: Description of Artificial Sequence: Degenerate PCR primer,12-F, used in the cloning of the partial cDNA of the hemolytic activeprotein of Carybdea rastonii <400> SEQUENCE: 8 gcngtngara aygcnaaymg 20<210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <221> NAME/KEY: unsure <222>LOCATION: (6) <223> OTHER INFORMATION: n = inosine <221> NAME/KEY:unsure <222> LOCATION: (15) <223> OTHER INFORMATION: n = inosine <221>NAME/KEY: unsure <222> LOCATION: (18) <223> OTHER INFORMATION: n =inosine <223> OTHER INFORMATION: Description of Artificial Sequence:Degenerate PCR primer, 12-R, used in the cloning of the partial cDNA ofthe hemolytic active protein of Carybdea rastonii <400> SEQUENCE: 9ckrttngcrt tytcnacngc 20 <210> SEQ ID NO 10 <211> LENGTH: 19 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (6) <223> OTHER INFORMATION: n = inosine <221>NAME/KEY: unsure <222> LOCATION: (15) <223> OTHER INFORMATION: n =inosine <223> OTHER INFORMATION: Description of Artificial Sequence:Degenerate PCR primer, 14-1-F, used in the cloning of the partial cDNAof the hemolytic active protein of Carybdea rastonii <400> SEQUENCE: 10gayggnttyt ayacnatgg 19 <210> SEQ ID NO 11 <211> LENGTH: 19 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (5) <223> OTHER INFORMATION: n = inosine <221>NAME/KEY: unsure <222> LOCATION: (14) <223> OTHER INFORMATION: n =inosine <223> OTHER INFORMATION: Description of Artificial Sequence:Degenerate PCR primer, 14-1-R, used in the cloning of the partial cDNAof the hemolytic active protein of Carybdea rastonii <400> SEQUENCE: 11ccatngtrta raanccrtc 19 <210> SEQ ID NO 12 <211> LENGTH: 23 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY:unsure <222> LOCATION: (6) <223> OTHER INFORMATION: n = inosine <221>NAME/KEY: unsure <222> LOCATION: (15) <223> OTHER INFORMATION: n =inosine <223> OTHER INFORMATION: Description of Artificial Sequence:Degenerate PCR primer, 12-2-F, used in the cloning of the partial cDNAof the hemolytic active protein of Carybdea rastonii <400> SEQUENCE: 12gayggnttyt ayacnatgga raa 23 <210> SEQ ID NO 13 <211> LENGTH: 23 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221>NAME/KEY: unsure <222> LOCATION: (9) <223> OTHER INFORMATION: n =inosine <221> NAME/KEY: unsure <222> LOCATION: (18) <223> OTHERINFORMATION: n = inosine <223> OTHER INFORMATION: Description ofArtificial Sequence: Degenerate PCR primer, 12-2-R, used in the cloningof the partial cDNA of the hemolytic active protein of Carybdea rastonii<400> SEQUENCE: 13 ttytccatng trtaraancc rtc 23 <210> SEQ ID NO 14 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: 5′Race primer, 5′-RACE-4R, synthesized based on the base sequence of thepartial cDNA for the hemolytic active protein of Carybdea rastonii <400>SEQUENCE: 14 gctctatcaa taacggcagc 20 <210> SEQ ID NO 15 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: 5′ Raceprimer, 5′-RACE-5R, synthesized based on the base sequence of thepartial cDNA for the hemolytic active protein of Carybdea rastonii <400>SEQUENCE: 15 tgtctttgga tggcctcatc 20 <210> SEQ ID NO 16 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: 5′ Raceprimer, 5′-RACE-6R, synthesized based on the base sequence of thepartial cDNA for the hemolytic active protein of Carybdea rastonii <400>SEQUENCE: 16 gatacttagg tcgctatccg 20 <210> SEQ ID NO 17 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: 3′ Raceprimer, 3′-RACE-1F, synthesized based on the base sequence of thepartial cDNA for the hemolytic active protein of Carybdea rastonii <400>SEQUENCE: 17 gttcagaggc tgttctaacg 20 <210> SEQ ID NO 18 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: 3′ RACEprimer, 3′-RACE-2F, synthesized based on the base sequence of thepartial cDNA for the hemolytic active protein of Carybdea rastonii <400>SEQUENCE: 18 atgtctgacg gcttctacac 20

What is claimed is:
 1. An isolated protein comprising the amino acidresidue sequence of SEQ ID NO:5 wherein said protein has hemolyticactivity.